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将人诺如病毒VA387株ORF2基因插入载体pFastBac1中,转化DH10Bac感受态细胞获得Bacmid-NoV-ORF2;脂质体介导转染sf9昆虫细胞,获得表达NoV-ORF2的重组杆状病毒Ac-NoV-ORF2.冻融破碎经Ac-NoV-ORF2感染的Tn5细胞,离心收集上清进行分子筛纯化.10%SDS-PAGE分析结果显示,重组病毒感染的Tn5细胞可见特异的蛋白条带;目的蛋白条带为相对分子质量59kD和56kD的两个条带;电镜观察发现表达的诺如病毒衣壳蛋白成功装配成了大小约为40~50nm的VLPs.结果表明在Tn5细胞中实现了诺如病毒衣壳蛋白的表达和VLPs的装配.
The ORF2 gene of Norovirus VA387 was inserted into pFastBac1 and transformed into DH10Bac competent cells to obtain Bacmid-NoV-ORF2. Lipofectamine was transfected into sf9 insect cells to obtain recombinant baculovirus Ac-NoV expressing NoV-ORF2 -ORF2.TN5 cells infected with Ac-NoV-ORF2 were thawed and thawed, and the supernatant was collected by centrifugation and purified by molecular sieve.The results of 10% SDS-PAGE analysis showed that specific protein bands could be observed in Tn5 cells infected with recombinant virus. With two bands of 59kD and 56kD, respectively.Electron microscopy showed that the expressed Norovirus capsid protein was successfully assembled into VLPs with the size of about 40-50nm.The results showed that Norovirus virus was achieved in Tn5 cells Expression of capsid proteins and assembly of VLPs.